Cholesterol homeostasis is maintained by balancing intestinal absorption, endogenous biosynthesis, hepatic uptake and biliary secretion of cholesterol and bile acids. Recently, there has been a renewed interest in the role of intestinal cholesterol absorption in cholesterol homeostasis. A number of studies have assessed the effect of reduced small bowel transit time on cholesterol absorption, and each found a significant correlation between transit time, cholesterol absorption and serum cholesterol concentration. We have demonstrated that mice with a targeted mutation in the inducible nitric oxide synthase (iNOS) gene have elevated serum cholesterol levels. Each of three unique isoforms of NOS is expressed in different cell types and under different conditions throughout the gastrointestinal tract. There is considerable evidence that nitric oxide (NO) produced in the gastrointestinal tract influences enteric myoelectrical activity, coordinates peristalsis and ultimately affects intestinal transit time. Nitric oxide acts as an inhibitory neurotransmitter, and as a vasodilator in the intestinal tract. Additionally, enteric pathogens, and the experimental administration of lipopolysaccharide (LPS), elicit excessive production of NO through transcriptional upregulation of iNOS. Thus, NO concentrations in the intestinal tract wax and wane depending on the immediate circumstances. A plausible explanation for the increased serum cholesterol levels observed in the iNOS-deficient mice, then, is delayed small bowel transit time, brought on by perturbation of the regulation and coordination of intestinal motility. Aberrant cholesterol homeostasis is a risk factor for both atherosclerosis and cholesterol gallstone formation. In addition to hypercholesterolemia, we observed aortic atheromas in the iNOS-deficient mice. Prolonged intestinal transit time augments formation of cholesterol gallstones, and if prolonged intestinal transit time is the underlying cause of hypercholesterolemia in iNOS-deficient mice, the manifestation of atherosclerosis and cholelithiasis may well be linked. The proposed studies will explore these possibilities in iNOS-deficient mice under various experimental conditions.